UP-REGULATION OF A NOVEL GENE BY FREEZING EXPOSURE IN THE FREEZE-TOLERANT WOOD FROG (RANA-SYLVATICA)

A novel gene responsive to freezing exposure was identified among five cDNA clones obtained through differential screening of a cDNA library constructed from liver of frozen wood frogs. The cDNA sequence of thi s gene, cloned in the recombinant plasmid, pBfFR14, showed no homology to any genes available in the Genbank database. The clone, designated as Fr10, carried a 457 bp cDNA sequence and contained a single open r eading frame that could potentially encode a small protein of 90 amino acids with a molecular weight of about 10 kDa, named FR10. The putati ve protein contained a highly hydrophobic N-terminal region (21 residu es) that carries a potential nuclear exporting signal (NES) sequence, LALVVLVIAISGL, similar to the NES found in PKI, an inhibitor of protei n kinase A (PKA). A single mRNA transcript with a size of 550 nt was d etected when the insert of the pBfFR14 was used as a probe against the Northern blot containing total RNA isolated from wood frog organs. RN A blotting analysis for gene expression in eight organs showed that tr anscription of the gene was highly induced by 24 h of freezing exposur e at -2.5 degrees C in liver and gut, moderately elevated in heart, lu ng, brain and bladder but showed no change in skeletal muscle and decr eased in kidney. A time-course analysis for freezing regulation of gen e expression in liver showed that transcript levels were increased by 2-fold in 1 h of freezing exposure and the levels continued to increas e up to 3.5-fold over the control after 24 h of freezing exposure, but had returned to control levels after 24 h thawing at 5 degrees C. Gen e expression in liver was also up-regulated by whole animal dehydratio n at 5 degrees C but strongly down-regulated by anoxia exposure, indic ating that the gene may respond to cell volume regulatory signals in v ivo during natural freezing. (C) 1997 Elsevier Science B.V.